The present invention relates to an electrochemical method and an associated microchip-based apparatus that can be used to afford voltammetric or amperiometric detection for monitoring immunochemical and/or molecular biology procedures.
To assess the-utility of any chemical reaction, whether it be inorganic, organic or biochemical, the composition and relative quantities of reactants and products must be determined while the reaction is in progress or at its equilibrium endpoint. One specific means of affecting such monitoring utilizes biologic or non-biologic molecules capable of binding to either reactant or product molecules in a structure restricted manner. These analytic techniques are, in general, referred to as immunochemical, in reference to the selective recognition and binding capacity of immunoglobulins, even though substances other than antibodies may serve as recognition molecules. The terms receptor and ligand have been used to more generally describe this area of analytic art. Although diversely applied in basic organic and biochemistry, these techniques have seen their most prolific development in the field of clinical medicine, and relevant generalized principles for measuring the progress of reactions immunochemically, although applicable to many other scientific pursuits, can be illustrated using examples from this field. Here, a multitude of immunochemically formatted tests have been developed for measuring virtually any biologic molecule of clinical importance. Such analytic procedures represent the cornerstones for laboratory studies in toxicology, endocrinology, immunology, serology, microbiology, and enzymology, to name but a few.
The most frequently utilized methods at present, are the enzyme-linked immunosorbant assays (ELISAs). These procedures are applicable to a wide variety of fields such as biotechnology, environmental protection and public health. The performance of these conventional state of the art calorimetric methods of detection suffer from the infirmities of having requirements for optical clarity, photomultiplication, signal digitalization or analog quantitation and transmission, viscosity or background chromogenic neutrality.
In the immunochemistry field, for example, enzyme immunoassays (EIA) and, more particularly enzyme-linked immunosorbent assays (ELISA) are well known in the art and have become important and relatively cost-efficient tools of clinical laboratories for detecting traces of foreign substances, such as antigens or antibodies in body fluids and tissues. (See, e.g. Immunoassay, Diamandis, E. and Christopoulos, T. eds., (1996); Clausen, J., Immunochemical Techniques for the Identification and Estimation of Macromolecules (Laboratory Techniques in Biochemestry and Molecular Biology) Vol. 1 (1989); Tijssen, P., Practice and Theory of Enzyme Immunoassays (Laboratory Techniques in Biochemistry and Molecular Biology), (1985); Principles and Practice of Immunoassay, 2d ed., Price, C. and Newman, D., eds. (1997)xe2x80x94which are hereby incorporated by reference in their entirety.)
Such immunoassays, while generally reliable, depend on sophisticated and extremely expensive optical processes to report their results. Such optical processes are cumbersome because they are expensive, require a clean and unsoiled measurement chamber and their visually rendered signals prevent precise quantitation of results in a simple manner. State of the art optical systems have several drawbacks, in that they generally require optical clarity, photo multiplication, signal digitalization or analog quantitation and transmission, as well as compatible viscosity and/or a neutral optical background. Transparent support media, aqueous or otherwise, may become fouled or turbid and prevent or render difficult any accurate analyses utilizing optical reporters.
Attempts have been made to provide systems other than optical ones to detect antigens in body fluids. Duan, C. et al., xe2x80x9cSeparation-Free Sandwich Enzyme Immunoassays Using Microporous Gold Electrodes and Self-Assembled Monolayer/Immobilized Capture Antibodies,xe2x80x9d Analytical Chemistry, 66/9:1369-77 (1994) discloses a separation-free system aimed at simplifying conventional immunoassay protocols utilizing a gold-plated microporous-membrane which serves as the solid phase for a noncompetitive sandwich-type immunoassay as well as a working electrode of an amperiometric detection system. A capture monoclonal antibody is covalently immobilized by a conventional chemical bonding agent at the gold plated side of the membrane. A model analyte protein as well as an alkaline phosphatase labeled antibody are incubated simultaneously with the immobilized capture antibody. Surface bound antibody is then separately detected from any excess conjugate in the sample by the introduction of an enzyme substrate, such as 4-aminophenol phosphate, from the backside of the membrane which is not gold-plated. The substrate seeps through the membrane and encounters the bound enzyme antibody conjugate at the gold-plated surface. Aminophenol is thus enzymatically generated and detected by oxidation at the gold electrode, the magnitude of the current being a measure of the concentration of analyte in the sample. However, the sensitivity of the system disclosed in Duan is very low, requiring a 20 nA signal compared to 0.1 nA in the present invention. This translates to a 50 times sensitivity advantage when considering actual protein detection limits. The system described by Duan was only capable of detecting protein (human chorionoic gonadotropin) down to a level of 500 ng/l, whereas the novel methodology herein described has shown a 10 ng/l protein detection limit.
In another experiment reported in Meyerhoff, M. et al., xe2x80x9cNovel Nonseparation Sandwich-Type Electrochemical Enzyme Immunoassay System for Detecting Marker Proteins in Undiluted Blood,xe2x80x9d Clinical Chemistry, 41/9:1378-1384 (1995), a similar microporous membrane was utilized in a non-separation sandwich-type electrochemical enzyme immunoassay system for detecting marker proteins in undiluted blood. However, this method is limited to prostate specific antigen (PSA) measurement in blood. The method described in this reference demonstrates no additional sensitivity when compared to the aforementioned article by Duan. Rather it simply describes the application of the technique to the measurement of an additional protein moiety (prostate specific antigen, PSA). Niwa, O. et al., xe2x80x9cSmall-Volume Voltammetric Detection of 4-Aminophenol with Interdigitated Array Electrodes and Its Application to Electrochemical Enzyme Immunoassay,xe2x80x9d Analytical Chemistry, 65:1559-1563 (1993) have reported on the use of an interdigitated array (IDA) micro-electrode cell in small-volume voltammetric detection of 4-aminophenol. However, Niwa used only alkaline phosphatase and used a sensor with a relatively small sensing area measuring 2xc3x972 mm and relatively large electrodes of width of 3 to 5 xcexcm, spaced 2 or 5 xcexcm from each other. Furthermore, their detection range was from 10 to 1,000 ng/ml for mouse IgG molecules above 1,000 nmol/l for p-aminophenol, which is about 100 times less sensitive than the present invention and does not make his technique viable for clinical applications with respect to disease-specific antibody detection and quantification.
H. T. Hang et al., in Anal. Him. Acta, 214:187-95 (1988) describes a system for the electrochemical detection of low molecular weight digoxin in the context of an immunoassay, but registers only currents generated by the oxidation of p-aminophenol.
Likewise in the molecular biology field, it is equally important to determine the composition and relative quantities of reactants and products while the reaction is in progress or at its equilibrium endpoint. One specific means of affecting such monitoring utilizes biologic or non-biologic labeling or reporter molecules capable of binding either reactant or product molecules in a structure-restricted manner. Many procedures commonly performed in the field of molecular biology fall into this category. Nucleic acid reactants or products have for many years been directly labeled by a variety of means such as the incorporation of radioactive 32-P or 3-H, or the use of electrophoretic gels incorporating intercalcating fluophores such as ethidium bromide. More recently, techniques have been borrowed from the immunochemical or receptor-ligand field and adapted to provide reporter systems that are safer, environmentally friendly, more cost effective, far faster, appropriate for use in a wide range of methods and compatible with efficiently conducting large numbers of procedures. Reporters have recently been introduced into the field of molecular biology that include detection by fluorescence, chemiluminescence, and colorimetry. These labels have been linked or conjugated directly to nucleic acid reactants or products, as well as generated indirectly via nucleic acid-enzyme conjugates in a manner comparable to ELISA techniques. (See, e.g., Tijssen, P., Hybridization With Nucleic Acid Probes: Theory and Nucleic Acid Probes, Vol. 1 (1993); Tijssen, P., Hybridization With Nucleic Acid Probes: Probe Labeling and Hybridization Techniques, Vol. 2 (1993); Meier, T. and Fahrenholz, F. eds., A Laboratory Guide To Biotin-Labeling in Biomolecule Analysis, BioMethods Vol. 7 (1996); Garman, A., Non-Radioactive Labelling: A Practical Introduction (Biological Techniques Series)(1997); Agrawal, S. ed., Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques (Methods in Molecular Biology, Vol. 26) (1993); Burden and Whitney, Biotechnology: Proteins to PCR: A Course in Strategies and Lab Techniques (1995)xe2x80x94which are hereby incorporated by reference in their entirety.) Detection of specific nucleic acid moieties using such reporters can be effectively performed while the reaction is in progress (rate measurement or kinetic measurement), or when the reaction has reached equilibrium (endpoint reporting). Molecular biology procedures using such reporter systems are commonly applied in many fields such as biotechnology, environmental protection and public health.
More specifically, recent advances in signal amplification methods (Dewar R. L., et al., xe2x80x9cApplication of Branched Chain DNA Signal Amplification to Monitor Human Immunodeficiency Virus Type 1 Burden in Human Plasma,xe2x80x9d Jrnl of Inf. Dis., Vol 170:1172-1179 (1994) as well as template amplification methods have resulted in a surge of nucleic acid detection and measurement techniques utilizing enzymatic conjugates in conjunction with calorimetric or chemiluminescent reporter products in place of the more hazardous, eco-unfriendly and temporally inefficient conventional radiographic reporters. All of these new reporters have, to present, relied on optical detection methods which, unfortunately, suffer from the infirmities of having requirements for solution clarity, photomultiplication, complex signal digitalization or analog quantitation and transmission, viscosity restrictions and requisite background chromogenic neutrality.
Therefore, despite all of these attempts at improvement, it is still the case that none of these systems describe an electrochemically detected enzyme-conjugate/reporter substrate capable of providing a cost effective method for direct testing of unprocessed immunochemical and biological samples with a satisfactory level of detection sensitivity.
It is the object of the present invention to provide an immunochemical and molecular biological reporter system to detect and quantify reactants or products. The present invention is intended to include both endpoint and kinetic reporting applications. This system consists of a silicon microchip-formatted interdigitated array (IDA) of closely spaced nobel metal electrodes used to detect immunochemical or nucleic acid conjugates containing electrochemically active molecules susceptible to redox recycling and therefore detectable by means of amperiometry or voltammetry.
It is a further object of the present invention to provide a system which can substitute for conventional calorimetric enzyme reporter systems to achieve enhancement relative to performance and economy.
It is a further object of the present invention to reduce the time necessary for completion of broad capacity for analytic procedures.
It is a further object of the present invention to increase the detection sensitivity relative to the absolute number of reporter molecules and the volume of solution required for their detection.
It is a further object of the present invention to expand the linear range of concentrations over which reporter molecules may be detected and quantitated.
It is a further object of the present invention to broaden the capacity for miniaturization and simplification of equipment relating to both methodologic and detection components supporting both hand-held as well as large-scale high throughput applications.
It is a further object of the present invention to eliminate the sample solution optical clarity or ambient optical density requirements.
It is a further object of the present invention to be able to use microliter or lower specimen requirements which correlates with reduced reagent costs.
It is a further object of the present invention to have manufacturing costs of IDA substantially in comparison to principle components photomultiplier requirements in comparable optical reporter systems.
In one preferred embodiment, this electrochemical reporter system may be applied to an immunochemical method for directing antibodies arising as a result of a viral infection by utilizing an immunoassay including a multivalent enzyme conjugate (Biotin/Avidin) for liberating redox-active molecules, and an IDA for measuring the redox-active molecules. In addition to the first embodiment, this novel reporter system is equally applicable to all enzyme-labeled immunochemistry formats. Methods to which this system applies are commonly, but not exclusively used to examine 1) Infectious diseases (microbial antigen or antibody proteins); 2) Autoimmune diseases (autoantigen or autoantibody proteins); 3) oncologic markers (so-called tumor specific proteins or steroids); 4) Endocrine hormones (polypeptides, thyronines and steroids); and 5) Therapeutic drugs or toxicologic materials.
In another preferred embodiment, this novel electrochemical reporter system may be applied to detecting or quantifying specific nucleic acids or their amplicons in analytic molecular biologic procedures. Analyses of specific nucleic acids or nucleic acid sequences are gaining wide acceptance and use in the clinical setting to examine body fluids or tissues for the presence of infectious microorganisms, malignancy, inherited disease (genetic defects), forensic medical evidence, and paternity/maternity identification.